Therapeutic modulation of the tumor inflammatory response

ABSTRACT

The invention relates to compositions, kits, and methods for alleviating cancer (i.e., a tumor) in a human patient. The therapeutic modality effected by the invention involves inducing a type 1 inflammatory response in the tumor tissue, whereby the tumor tissue is diminished or destroyed and the patient develops immune memory that inhibits or prevents recurrence of the tumor.

CROSS-REFERENCE TO RELATED APPLICATIONS

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STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

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REFERENCE TO A MICROFICHE APPENDIX

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BACKGROUND OF THE INVENTION

Human (and other vertebrate) immune responses comprise complex andintricately regulated sequences of events, involving cells of severaldifferent types. An immune response can be triggered when an antigen ina human body encounters an antigen-presenting cell (APC). The APC cancapture the antigen and display a portion of it on its surface in a formthat can be recognized by a helper T (Th) lymphocyte. Upon binding of aTh lymphocyte with the APC-displayed antigen, the Th lymphocyte canbecome activated. An activated Th lymphocyte promotes activation oflymphocytes of other types, the particular type(s) depending on theidentity of the Th lymphocyte and the context in which the antigen isdisplayed by the APC. Various types of Th lymphocytes can, for example,promote activation of cytotoxic T cells or proliferation ordifferentiation of antigen-specific B cells. Th lymphocytes can activateother lymphocytes by secreting one or more polypeptide hormonesdesignated cytokines. Th lymphocytes exhibit the CD4 antigen on theirsurface.

Functionally distinct types of Th cells have been described in humans.At least two types of Th cells have been characterized, based on thecytokines they produce. Type 1 Th cells (i.e., Th1 cells) can produceinterleukin-2 (IL-2), interferon-gamma (IFN-g), and tumor necrosisfactor-beta (TNF-b). Type 2 Th cells (i.e., Th2 cells) can produce IL-4,IL-5, IL-6, and IL-10.

Immune responses activated by Th1 cell activation (i.e., a ‘type 1immune response’ or ‘Th1-mediated response’) are characterized bysignificant production of IFN-gamma and promotion of cytotoxiclymphocyte activity. Type 1 immune responses can be induced, forexample, by the presence of bacteria in the human body. The cytotoxiclymphocytes activated in a Th1-mediated response are capable ofrecognizing and killing cells that display the Th1-activating antigen ontheir surface. Thus, induction of a type 1 immune response can lead toelimination from the body of antigen-bearing cells. Excessive orotherwise inappropriate induction of a type 1 immune response can causedamage to normal (i.e., non-diseased tissues) in a human.

Immune responses activated by Th2 cell activation (i.e., a ‘type 2immune response’ or ‘Th2-mediated response’) are characterized bysignificant production of IL-4 and promotion of humoral immunity (e.g.,production of immunoglobulins, particularly including IgE). Type 2immune responses are commonly induced in response to chronic infections(e.g., parasitic infections), and tend to inhibit, prevent, or reversetype 1 immune responses. Type 2 immune responses will normally eliminatea pathogen from the body, and can thereby inhibit further infection byan infectious agent. However, type 2 immune responses generally do noteliminate all pathogen-infected or diseased cells from the body. Thus,cells which exhibit the antigen that induced the type 2 immune responsemay persist chronically in the body. This occurs particularly whenantigen-bearing cells (e.g., virus-infected cells or tumor cells) induceinappropriate activation of a type 2 immune response, which canfacilitate persistence of the antigen-bearing cells in a human body.

Inflammation is a normal localized immune response to invasion or injurycaused by an infectious agent (e.g., a bacterium) or by a tumor. In aprocess analogous to the manner in which blood flow can increase thesupply of glucose and oxygen to active muscle tissue during a period ofexercise, an inflammatory response can increase the supply of elementsof the immune system at a local disease (e.g., infection or tumor) sitein order to mount an effective defensive immune response. An effectiveinflammatory response can be characterized by at least six events:

i) release of antigens from diseased or pathological cells at thedisease site and secretion of chemotactic factors at the injured site;

ii) infiltration of the disease site by cells of the immune system;

iii) polarized type I or type II activation of the immune cells by theantigens released at the site;

iv) amplification of the inflammatory response over time, at least for alimited period;

v) elimination of the diseased or pathological cells by immune cells;and

vi) conversion of activated immune cells into memory cells which arecapable of providing long-term protection against the antigen orantigen-bearing cells.

Cancer is one of the foremost causes of mortality and morbidity amonghumans. Many cancers are manifested by the existence of tumors, whichare clumps or masses of cancer cells. In the past, cancer cells weregenerally believed to be non-immunogenic, since they are derived fromautologous (i.e., ‘self’) tissue, which normally does not induce animmune response. However, tumor-reactive lymphocytes can be isolatedfrom patients afflicted with many types of cancer (Lee et al., 1997,Blood 90:1611-1617).

A significant portion of tumor mass is made up of lymphocytes. Thesecells, designated tumor-infiltrating lymphocytes (TILs), typicallyproduce cytokines (e.g., IL-4) that are characteristic of a type 2inflammatory response (Roussel et al., 1996, Clin. Exp. Immunol.105:344-352). It has been postulated that predominance of type 2 TILssupport a type 2 inflammation in tumors that inhibits tumoricidalcytotoxic immune responses. It has furthermore been suggested thatmodulation of the type of immune response exhibited by TILs can haveanti-cancer therapeutic effects (Gorelik et al., 1994, Cancer Immunol.Immunother. 39:117-126; Pellegrini et al., 1996, Cancer Immunol.Immunother. 42:1-8; Goedegebuure et al., 1997, Cell Immunol.175:150-156; Fujimoto et al., 1997, J. Immunol. 158:5619-5626; Okamotoet al., 1997, Int. J. Cancer 70:598-605; Stein et al., 1998, Eur. J.Med. Res. 3:194-202; Li et al., 1998, J. Surg. Oncol. 67:221-227).However, despite this recent understanding regarding both types ofinflammatory response, no course of treatment has previously beenidentified whereby a type 2 inflammatory response (i.e., one conduciveto tumor survival or growth) can be converted to or overcome by a type 1inflammatory response (i.e., one in which tumor growth slows or haltsand tumor regression is enhanced).

Current cancer therapies (e.g., surgery, radiotherapy, and chemotherapy)are relatively inefficient, and have very debilitating side effects thatlead to relapses and death. In view of the overwhelming toll of humanmortality and morbidity associated with cancer, an urgent need remainsfor therapeutic compositions, kits, and methods which can slow orreverse tumor progression in humans while reducing morbidity andoffering protection against tumor relapse. The present inventionsatisfies this need, at least in part, by providing therapeuticcompositions, kits, and methods which can be used to reliably treat avariety of human cancers, reduce treatment-related morbidity (relativeto prior art therapeutic methods), and offer protection against tumorrelapse.

BRIEF SUMMARY OF THE INVENTION

The invention relates to a method of alleviating a tumor in a humanpatient. The method comprises locally administering to the tumor (i) anantigen-releasing agent, (ii) a leukocyte attractant, and (iii)interferon-gamma (IFN-g) and a second type 1 inflammatoryresponse-(IR1-)promoting agent (e.g., one of linterleukin-2 (IL-2),interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF-a), and tumornecrosis factor-beta (TNF-b)).

The antigen-releasing agent induces release of one or more tumorantigens from cells of the tumor, and can, for example, be one of aproteolytic enzyme, an apoptosis-inducing agent, electrical current, astrong acid, and a strong base. The leukocyte attractant inducesleukocytes to infiltrate the tumor. Together, the agents administered tothe tumor induce a type 1 inflammatory response in the tumor andalleviate the tumor (i.e., cause it to shrink or disappear). An addedbenefit of this therapeutic method is that the incidence of tumorrecurrence can be decreased, relative to other tumor alleviationmethods.

In one embodiment, the method further comprises locally administering tothe tumor a type 1 lymphocyte attractant (e.g., one of RANTES, IP-10,and Mig), in order to sustain the type 1 inflammatory response. The type1 inflammatory response can also (or instead) be sustained byadministering autologous leukocytes to the patient. These leukocytes canbe isolated from the patient, expanded ex vivo, induced todifferentiate, and returned to the patient, preferably by localadministration at the tumor site

In order to reduce recurrence of the tumor, a memory cell-inducing agent(interleukin-15 (IL-15) or interferon-alpha (IFN-a)) can be administeredto the patient in order to enhance production of anti-tumor type 1immune memory cells. When used, the memory cell-inducing agent ispreferably administered locally to the tumor site after most (e.g., 90%)of the tumor mass has disappeared, but before the tumor has beencompletely ablated.

The therapeutic method can further comprise supplementing the patient'snutrition with a nutrient such as a vitamin (e.g., one or more ofvitamins A, B, C, D, and E) or a mineral (e.g., one or more of selenium,zinc, calcium, magnesium, iron, and copper).

The invention also includes compositions useful in performing a tumortherapeutic method described herein. For example, such a composition cancomprise IFN-g and a second IR1-promoting agent. Local administration ofthe composition to a tumor induces a type 1 inflammatory response in thetumor, and the tumor is thereby alleviated. The composition can furthercomprise one or more of a leukocyte attractant, an antigen-releasingagent, and a pharmaceutically acceptable carrier.

The invention also includes a kit for alleviating a tumor in a humanpatient. The kit can comprise one or more of the agents used in themethods described herein, equipment and devices used in those methods,and an instructional material which describes one or more of themethods.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compositions, kits, and methods for alleviationof cancer in human patients. The invention is based on inducing a type 1inflammatory response at the site of a tumor. Induction (or enhancement)of a type 1 inflammatory response at a tumor site (i.e., within and nearthe margins of a tumor) can cause the tumor to shrink or disappear,reduce the invasiveness or metastatic capacity of the tumor, and enhanceproduction of immune memory cells which specifically recognize tumortissue and inhibit or prevent relapse (i.e., recurrence) of the tumor.

Although the desirability of exchanging a type 1 immune response intumor tissue in place of the normally-prevalent type 2 response isbecoming better understood, there is presently no treatment for reliablyestablishing a type 1 immune response in tumor tissue. The presentinvention overcomes the deficiencies of the prior art by providingtherapeutic compositions, kits, and methods which can reliably be usedto activate Th1 cells in and around tumor tissue, with the result thattumor-cytotoxic inflammation is induced in the tumor, leading to itsregression or elimination.

Prior art methods of inducing tumor cell death include administration ofagents which are cytotoxic to tumor cells, but which either exhibitlesser cytotoxicity with respect to non-tumor cells or are selectivelydelivered to tumor cells. Although these methods have exhibited limitedsuccess at limiting the size and rate of progression of tumors, at leastfor limited periods, many of these methods also exhibit a criticaldrawback that limits their effectiveness. Delivery of a cytotoxic agent(e.g., radiation or a cytotoxic chemical compound) to a tumor can killnot only tumor cells, but also any non-tumor cells which are present inthe tumor. This cytotoxicity can be caused by the direct effect of theagent on the non-tumor cells, by a ‘by-stander’ effect wherein the agentinduces localized cytotoxicity that is not specific for tumor cells(e.g., by release of a cytotoxin such as ricin from an agent intended todeliver the cytotoxin specifically to tumor cells), or by othermechanisms. Tumors normally comprise a significant number of leukocyteswhich, if appropriately activated, can induce tumor cell death and leadto tumor regression. Many anti-tumor cytotoxic agents kill theselymphocytes in addition to tumor cells. Thus, although many prior artanti-tumor agents kill significant numbers of tumor cells, the agentsalso kill significant numbers of leukocytes in the tumor. Elimination orinactivation of these tumors reduces the body's ability to marshal itscytotoxic immune resources to combat the tumor. The net effect ofanti-tumor treatment using many of these prior art anti-tumor agents istemporary reduction in tumor mass without enhancement of the body'sability to destroy the tumor remnants or prevent recurrence of thetumor. As a result, tumor recurrence frequently occurs following priorart anti-tumor treatment methods.

The methods described in this specification can be distinguished fromprior art anti-tumor therapeutic methods in several ways. For example,the methods described herein enhance activity and proliferation of thebody's anti-tumor cytotoxic immune cells within and in the immediatevicinity of the tumor, rather than killing these cells as in prior artmethods. Furthermore, by enhancing activity and proliferation ofanti-tumor immune cells, the methods described herein can inhibit orprevent recurrence of the tumor in that patient's body.

The anti-tumor therapeutic methods described herein can be outlined asfollows. Enzymes or other compounds which cause release of antigens fromtumor cells (and which preferably also induce tumor cell death) aredelivered locally to a tumor. These compounds are selected such thatthey are not cytotoxic with respect to lymphocytes (or at least lesscytotoxic with respect to lymphocytes than with regard to tumor cells).TILs are attracted to the tumor site by local administration of one ormore chemokines to tumor tissue. Local administration of type Ilymphokines to the tumor polarizes the TILs to exhibit a type 1inflammatory response, leading to tumor tissue destruction. Sustainedamplification of the type 1 inflammatory response can be effected byrepeated local delivery of the type 1-polarizing cytokines to the tumorsite. The net effect of this treatment is that the body's own immunedefenses are mobilized for destruction of the tumor. Continued survivalof type 1-polarized lymphocytes, converted into memory cells, in thepatient's body can prevent recurrence of the tumor at the same site orat a different body location. These lymphocytes can also travel to anddestroy related tumors which may exist in the patient's body (e.g., bymetastasis of cells from the original tumor).

These methods, and compositions and kits for performing them, are setforth in greater detail in the following sections.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

A “tumor” refers to a solid or semi-solid mass of tissue comprisingcells which exhibit uncontrolled growth characteristic of cancer. Tumorsinclude both benign tumors (i.e., those which do not appear to beinvading surrounding tissues or metastasizing to other body sites) andmalignant (i.e., non-benign) tumors.

“Co-administration” of two or more agents means administration of theagents sufficiently close in time that the periods of significantactivity of each of the agents in vivo overlaps. By way of example,co-administration includes administration of a first and a second agentsufficiently close in time that the level of activity, in vivo, that isattributable to the first agent is more than half its maximumpost-administration level at a period of time when the level ofactivity, in vivo, that is attributable to the second agent is more thanhalf its maximum post-administration level. Co-administered agents canbe administered in the form of a single composition comprising eachagent, in the form of individual agent preparations, or (i.e., whenthere are more than two agents) in combinations of such forms.

Description

Tumorigenesis is a largely continuous occurrence in animals such ashumans. Every day, millions of cell divisions occur in a human, andmutations and other genetic lesions inevitably occur in at least some ofthese divisions, leading to generation of cells which exhibituncontrolled growth (i.e., cancer cells). Recognition and destruction ofcancer cells are among the normal functions of the human immune system.When appropriately activated, cells of the type 1 immune response(including, for example, cytotoxic T cells) are capable of specificallydestroying cells which exhibit abnormal cell surface markers (e.g.,cancerous or virus-infected cells). However, survival of cancer cells issometimes promoted by activation of cells of the type 2 immune response,which are normally associated with inactivation of parasitic or chronicinfective agents, but which are not normally associated withtumorotoxicity. The type 2 immune response can sometimes be induced bythe cancerous cells themselves, such as when the cancerous cells secretefactors which promote induction of the type 2 immune response.

Among the characteristics of the type 2 immune response is that type 1inflammatory responses are inhibited when it occurs. Because tumoricidalcytotoxicity is associated with the type 1 inflammatory response, butnot with the type 2 immune response, the type 2 immune state can prolongsurvival of tumor tissue and permit that tissue to evade the patient'snormal immune defenses.

The anti-cancer therapeutic method described in this specificationinvolves altering the type 2 immune response which normally occurs intumor tissue such that a type 1 inflammatory response takes over (i.e.,type 1 inflammation is initiated or de-repressed). Cells associated withthe type 1 inflammatory response recognize and kill tumor cells, therebyachieving tumor reduction (or even elimination) in the absence of theoften debilitating or disfiguring side effects associated withadministration of prior art anti-tumor agents.

In a preferred embodiment, the treatment method comprises threeelements, as set forth in the three ensuing paragraphs. Although theelements are designated ‘first,’ ‘second,’ and ‘third,’ the actionscorresponding to these elements need not be performed in this sequentialorder (although, in a preferred embodiment, the actions corresponding tothe first element are performed prior to {e.g., at least two hours priorto} the actions corresponding to the second and third elements).

First, one or more agents (e.g., tumor de-bulking agents such asproteases or mixtures of proteases) are administered directly to thetumor tissue (e.g., by injection into the tumor at one or more sites) inorder to cause release of tumor antigens from tumor cells. Without beingbound by any particular theory of operation, it is believed that releaseof antigens from tumor cells permits those antigens (e.g., fragments oftumor cell surface proteins) to interact with immune cells in ways thatare not possible when the antigens remain part of a tumor cell. Releaseof tumor antigens from tumor cells facilitates induction of immuneresponses which are specific for the tumor cells.

Second, TILs are recruited to the tumor site by local administration ofone or more TIL chemoattractants. TILs that are recruited to the tumorsite can be activated to secrete chemokines which attract more TILs,induce the TILs to exhibit a type 1 inflammatory response, or both.

Third, leukocytes present at (or recruited to) the tumor site areinduced to exhibit a type 1 inflammatory response by locallyadministering two or more type 1 inflammatory response promoting agents(“IR1-promoting agents”) including interferon-gamma (“IFN-g”) withothers such as linterleukin-2 (“IL-2”), and tumor necrosis factor beta(“TNF-b”) to the tumor tissue. The IR1-promoting agents are preferablyadministered together in a single composition; however, they can beadministered separately, but closely in time (e.g., seconds or minutesup to a few hours apart). The IR1-promoting agents can be locallyadministered to the tumor, for example, by injecting them into one ormore sites in the tumor, peritumorally, or both. Preferably, theIR1-promoting agents are injected into two or more sites in the tumor,the sites spaced approximately equally apart in the tumor mass.

In the presence of two or more of these agents, TILs exhibitcharacteristics of a type 1 inflammatory response, such as the abilityto kill tumor cells and virus-infected cells. Such TILs can destroytumor tissue, leading to shrinkage, or even disappearance, of the tumorfrom the patient's body and alleviation of tumor-related symptoms (e.g.,pain, weight loss, nausea, exhaustion, and symptoms associated with thepresence of a tumor at a particular body location).

Although the tumor treatment method can include only these three steps,preferred embodiments of the method include one or more of threeadditional steps, which are designated the fourth, fifth, and sixthsteps in the ensuing three paragraphs.

Fourth, the effectiveness (i.e., sustained potency) of the treatment canbe enhanced by amplifying the type 1 inflammatory response induced inand/or around the tumor tissue. This can be achieved, in one embodiment,by additional (e.g., sustained or repetitive) local administration ofthe same or different IR1-promoting agents to the tumor tissue or thetumor site. Alternatively, or in addition, autologous lymphocytes can beprovided to or near the tumor site, in order to provide a greater poolof lymphocytes from which TILs can be generated or separated. Theautologous lymphocytes can have been induced to proliferate ex vivo, inorder to enhance the number of cells available for administration to thetumor site. The additional lymphocytes can also have been induced todifferentiate into Th1-VLA6⁺ or Th1-CD49f⁺ cells by treating them, exvivo, with one or more IR1-promoting agents.

Fifth, generation, proliferation, or both, of immune memory cells can beinduced by injecting a memory cell-inducing agent (e.g.,interferon-alpha {“IFN-a”} or interleukin-15 {IL-15}) at or near thetumor site at one or more steps of the treatment. Immune memory cells(e.g., activated B or T lymphocytes) are normally formed in the presenceof an antigen, and normally are able to rapidly differentiate to formtype 1 and type 2 immune cells upon re-exposure to the antigen. However,during a type 2 immune response, formation of type 1 immune memory cellscan be inhibited and their differentiation or activation in response tothe presence of the antigen can be inhibited. Formation of these type 1memory cells is preferably enhanced during the period of type 1inflammatory response brought about by the other steps of the treatmentdisclosed herein, resulting in formation of type 1 memory cells whichcan rapidly respond to recurrence of the tumor at the same or adifferent body site.

Sixth, the effectiveness of the anti-tumor treatment disclosed hereincan be enhanced by enhancing the patient's general state of health, andparticularly by enhancing the state of the patient's immune system.Nutritional supplementation methods are known in the art, and preferredregimens are described herein.

In the following sections, particular aspects of these six steps aredisclosed. The anti-tumor treatment includes at least the steps ofenhancing tumor antigen release, inducing tumor infiltration bylymphocytes, and induction of a type 1 inflammatory response. Of course,two or more of these steps can be combined, although the antigen-releasestep preferably precedes the others by a period of at least severalhours (i.e., in order to generate a gradient of tumor antigen having itsfocus at the tumor site). One or more of the fourth, fifth, and sixthsteps described above (and preferably all three) can be included in themethod. The sixth step can be performed at any time during the method,and is preferably performed throughout. The fourth and fifth stepspreferably follow the antigen-release step by a period of at leastseveral hours.

Enhancing Tumor Antigen Release

The method includes inducing release of antigens from tumor cells. Manymethods which induce antigen release are also associated withsignificant cytotoxicity. Thus, the antigen release step can effectde-bulking of the tumor as well. So long as more (preferablysignificantly more) tumor cells than lymphocytes are killed, thede-bulking can enhance or speed the anti-tumor treatment by reducing thetumor burden in the patient. Tumor de-bulking can be achieved byadministering a tumor de-bulking agent to the tumor in order to causedeath of at least a fraction of tumor cells and release of tumorantigens from the killed tumor cells. The antigen-release-inducing agentcan be administered locally to the tumor tissue, systemically, or in anyother manner in which the agent is brought into contact with the tumortissue.

The antigen-release-inducing agent is preferably one which exhibitsrelatively little or no cytotoxicity with regard to leukocytes,particularly with regard to TILs. Examples of suitableantigen-release-inducing agents include compositions comprising one ormore de-bulking agents, which include proteolytic enzymes such astrypsin, chymotrypsin, pepsin, and collagenase, apoptosis-inducingagents such as alkylphospholipids (e.g., alkylphosphocholines such ashexadecylphosphocholine or edelfosine), electrical current (e.g.,delivered by way of electrodes inserted into tumor tissue), and strongacids and bases (e.g., concentrated solutions of sodium and potassiumhydroxides and hydrochloric acid). The agent is administered locally tothe tumor tissue (e.g., by topical application to the tumor tissue or byintratumoral and/or peri-tumoral injection) in order to decrease damageto non-tumor tissues.

Surgical tumor disruption and radiative tumor disruption methods can beused to induce release of antigens from tumor cells. However, thesemethods are not preferred, owing to their relative non-specificity withrespect to killing tumor cells and lymphocytes. Of course, it can bepossible to overcome this limitation by providing lymphocytes to thetumor site from an extracorporeal source (e.g., autologous lymphocyteswhich have been induced to proliferate, differentiate, or both, exvivo).

The following four paragraphs describe examples of methods of inducingrelease of antigens from a tumor located in a patient's body.

An aqueous composition comprising one or more proteases (e.g., one ormore of trypsin, chymotrypsin, pepsin, and collagenase) is prepared,wherein the concentration of each protease is about 2,000 to 10,000units per cubic centimeter of tumor volume to be targeted. An aliquot ofthis solution representing from about 1/50 to 1/100 of the volume of thetumor to be affected is injected into the tumor. For example, about 100microliters of a suspension of several proteases, each having aconcentration of about 50,000 units per milliliter can be injected intoa tumor having a volume of about 10 milliliters. This solution ispreferably injected into several locations in the same tumor, in orderto distribute the antigen-releasing and/or tumor-de-bulking effectsthroughout the tumor mass. Using this treatment, about 10-20% of thetumor can be expected to be digested (i.e., about 10-20% of tumor cellswill be killed). Of course, other concentrations and combinations ofenzyme can be used.

In another embodiment, antigen release from a tumor is induced byinjecting a solution of one or more alkylphosphocholines into the tumormass, preferably at spatially distinct locations. Alkylphosphocholinesare phospholipids which have structural similarity tonaturally-occurring cell membrane phospholipids. Alkylphosphocholinessuch as hexadecylphosphocholine and edelfosine(1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) are relativelynon-toxic with regard to lymphocytes, but can induce disruption (i.e.,apoptosis) of tumor cells, thereby effecting antigen release. Asuspension containing about 50-250 milligrams of eachalkylphosphocholine per milliliter of tumor volume can be prepared, andan aliquot having a volume of about 1/50 to 1/100 the tumor volume canbe injected. For example, 200 microliters of a suspension comprising 25%(w/v) hexadecylphosphocholine or edelfosine can be injected into a tumorhaving a volume of about 10 milliliters. The treatment described in thisparagraph can be expected to disrupt about 10-20% of the tumor (i.e., toinduce apoptosis of 10-20% of tumor cells).

In another embodiment, release of antigens from tumor cells can beinduced by applying electrical potential across a portion of the tumor(i.e., by passing electrical current through a portion of the tumor).Electrochemical tumor disruption can be effected by inserting anelectrode having a polarity (e.g., an anode) in one portion of a tumormass, inserting an electrode having the opposite polarity (e.g., acathode) in another portion of the tumor mass, and applying anelectrical potential across the electrodes. Of course, multipleelectrodes of each type can be used, so long as there is at least oneelectrode having a polarity opposite to that of another electrode. Forexample, an anode having a diameter of about 0.5 millimeter can beinserted in about the center of a tumor mass, and multiple cathodes canbe inserted into the periphery of the tumor mass, about 3 centimetersapart from each other. A direct current potential of about 5-10 Volts isapplied between the anode and cathodes, and about 40-100 milliamperes ofcurrent are delivered for a period of about 1-2 hours. The total chargedelivered to the tumor during this period is estimated to be about 60-80Coulombs per cubic centimeter of tumor. This treatment can be expectedto induce tumor tissue destruction within about 3 centimeters from eachelectrode.

Disruption of tumor tissue and release of tumor antigens can also beeffected by direct administration to tumor tissue of a solution ofconcentrated acid or base. For example, a 10 molar solution ofhydrochloric acid or a 10 molar solution of sodium hydroxide can beadministered intra-tumorally. An aliquot of either of these solutionsequal in volume to about 1/50 to 1/100 of the tumor volume can beinjected (for example, 100 microliters of one of these solutions can beinjected into a tumor having a volume of 10 milliliters). The solutionis preferably injected into two or more spatially distinct locations inthe tumor mass, although a single injection can be used for smalltumors. Treatment as described in this paragraph can be expected toinduce death of about 10-20% of tumor cells, and the concentration oridentity of the acid or base can be adjusted to effect this level oftumor cell killing.

Inducing Tumor Infiltration by Lymphocytes

The anti-tumor therapeutic method includes a step wherein leukocytes areattracted to the tumor site, or into the tumor itself. Recruitment ofleukocytes at a particular location can also be effected by causingleukocytes that are already at the location to proliferate. It is knownin the art that leukocytes of particular types can be attracted to alocation in a body (or in vitro) by the existence of one or moreparticular chemokines at the location. Of course the particular types ofleukocytes vary with the particular chemokines. For example, monocytescan be attracted to a site by the presence of MCP-1, MCP-2, MCP-3, orMCP-4. T cells are attracted by RANTES, IP-10, or Mig. Eosinophils canbe attracted by the presence of eotaxin at a body location. Furthermore,recruitment of type 1 inflammatory cells can be enhanced by inducingproliferation or activation of the cells at the site. For example, IL-2is known to induce proliferation of lymphocytes which exhibit the CD4antigen (including Th1 lymphocytes) and to induce activation oflymphocytes which exhibit the CD8 antigen (e.g., cytotoxic Tlymphocytes).

Leukocyte infiltration into a tumor is preferably induced following aperiod of several (e.g., 6-8) hours following induction of tumor antigenrelease. Waiting for this period prior to inducing leukocyteinfiltration permits conditions used to disrupt tumor cells, or induceantigen release therefrom, to dissipate. This period also provides timefor a gradient of tumor antigen to develop having the tumor at its mostconcentrated location. Formation of such a gradient can enhance theability of lymphocytes and macrophages to localize specifically withinthe tumor.

One embodiment of a method by which infiltration of lymphocytes into atumor is induced is as follows. A suspension comprising IFN-g and TNF-ais prepared, comprising about 20-100 units per milliliter of tumorvolume of IFN-g and about 100-500 units per milliliter of tumor volumeof TNF-a. The suspension further comprises IP-10 and Mig, each at aconcentration of about 1-100 nanograms per milliliter of tumor volume.An aliquot of this suspension is administered intra-tumorally, thevolume of the aliquot being approximately 1/50 to 1/100 the volume ofthe tumor. The aliquot is preferably administered by injection into oneor more sites within the tumor, multiple sites preferably beingapproximately equidistant from one another. Injection of this suspensionactivates monocytes such that they produce monocyte chemoattractants andalso attracts TILs to the tumor. In some instances, IP-10, Mig, or both,can be omitted from the suspension, because these TIL chemoattractantsare normally produced by activated monocytes (which are attracted to thetumor site by IFN-g and TNF-a). The suspension can be repetitivelyadministered to the tumor, or administered in a sustained-releaseformulation; however, repetitive and sustained administration will oftenbe unnecessary, owing to the self-sustaining nature of the type 1inflammatory response induced by attracting monocytes to the tumor site(i.e., the monocytes attract more monocytes and TILs).

In another embodiment, granulocytes (e.g., neutrophils and basophils)are attracted to the tumor site by including one or moregranulocyte-attractive agents in the suspension of chemokines that isadministered to the tumor. Examples of such agents include IL-8,granulocyte chemotactic protein-2 (GCP-2), growth-related oncogens 1, 2,and 3 (GROs), neutrophil-activating protein 2 (NAP-2), and others knownin the art. The desirability of attracting granulocytes to a tumor candepend on the type of tumor being treated. For example, some tumors(e.g., certain brain tumors, such as gliomas) do not form solid tissuemasses, but instead have a gelatinous consistency. In gelatinous tumors,granulocytes can enhance inducement, endurance, or both, of a type 1inflammatory response in the tumor tissue, presumably attributable tocytotoxic factor exhibited by granulocytes. Thus, it can be preferred toinclude administration of a granulocyte-attracting chemokine in acomposition, kit, or method for treating a semi-solid tumor.

In addition, it is recognized that certain chemokines can be moreconveniently provided to certain tissues than others. For example, thechemokine designated neurotactin (sometimes designated fractalkine ortype 1 membrane protein) can be administered to brain tissue (i.e., fortreatment of brain tumors).

The effectiveness of numerous individual chemokines for inducinginfiltration of lymphocytes into tissues of various types is known,although not all of these characteristics are replicated in thisspecification.

Initiation of the type I inflammatory response can, alternatively, beachieved or supplemented by injecting another inflammation-inducingagent into the tumor mass. For example, modified bacteria (e.g.,Bacillus Calmest-Guerin), lipopolysaccharides, or mild tumor-de-bulkingor tumor-antigen-releasing agents can be used to induce inflammationthat will attract monocytes to the site. Once attracted to the site, themonocytes can secrete Th1-attracting lymphokines. Because it ispreferable to achieve rapid and efficient induction of a type 1inflammatory response, administration of IP-10, Mig, or (preferably)both, is the preferred method of inducing inflammation.

Inducing a Type 1 Inflammatory Response

The anti-tumor therapeutic methods described in this specification alsoinclude a step in which the lymphocytes present within a tumor or at thetumor site are induced to exhibit a phenotype and functional propertiescharacteristic of a type 1 inflammatory response. Activation of Th1cells and T cytotoxicl (Tc-1) cells which have cell surface receptorsthat bind specifically with tumor antigens is desirable, and contributesto tumor cell cytotoxicity. In some embodiments of the method,granulocytes can also be activated such that they contribute to the type1 immune response and contribute to tumor cell cytotoxicity.

Promotion of a type 1 immune response within or in the vicinity of atumor can be achieved by local administration of lymphokines which arenormally produced by lymphocytes associated with a type 1 immuneresponse. For example, a suspension of IL-2, IFN-g, and TNF-b can beadministered in order to achieve this effect. The suspension can alsocomprise TNF-a and IL-12. Any combination of these five lymphokines canbe used. However, the combination of IL-2, IFN-g, and TNF-b ispreferred, because it has been discovered that this combination exhibitsbetter synergy and permits use of lower doses of each of the threeagents. For example, one useful suspension comprises 10-100 units ofIL-2 per milliliter of tumor volume, 100-1000 units of IFN-g permilliliter of tumor volume, and 50-500 units of TNF-b per milliliter oftumor volume. An aliquot of this suspension having a volume from 1/50 to1/100 of the volume of the tumor to be treated is administeredintra-tumorally, at one or more sites in the tumor mass. The lymphokinesuspension should be administered every 48-72 hours until the tumor massbecomes very small (e.g., less than 5% its original size) orundetectable (e.g., using digital imaging techniques such as CATscanning). Alternatively, the lymphokine composition can be delivered ina sustained release form, so that fewer repetitions of theadministration are necessary. For example, slowly-dissolving rodscomprising a biodegradable matrix (e.g., PLGA) having the lymphokine(s)sequestered therein can be used to effect delivery over a period of daysor weeks.

Sustained Promotion of Type 1 Inflammatory Response

The effectiveness of the anti-tumor therapeutic method described in thisspecification can be enhanced by multiple (or sustained or continuous)local administration of one or more chemokines, followed by localadministration of cytokines normally associated with a type 1 immuneresponse. The effectiveness of the method can also be enhanced by singleor multiple provision of leukocytes expanded or differentiated in vitro.

For example, Mig and IP-10, two chemokines that attract both Th1 and Tc1lymphocytes are prepared in a suspension comprising 10-500 nanogramseach per milliliter. This suspension can be injected into the tumor lessthan about an hour before administering autologous lymphocytes that havebeen expanded or differentiated in vitro. Providing these chemokines todiffuse in the tumor before providing the lymphocytes enhancesinfiltration of the lymphocytes into the tumor. Without being bound byany particular theory of operation, this enhancement of infiltration isbelieved to be attributable to the ability of these chemokines toactivate avidity of the beta-1 integrin receptors on lymphocytes,causing them to migrate along the chemokine gradient. Thus, othercompounds having this same effect can be used in place of thesechemokines.

Lymphocyte populations can also be expanded and differentiated in vitrousing known methods. Preferably, the lymphocytes used in such in vitroprocedures are obtained from the patient to whom they expanded ordifferentiated lymphocytes are to be administered, so that the patient'simmune system will not reject the lymphocytes and so that theadministered lymphocytes will not attack healthy (e.g., non-tumor)patient tissues. Also, the lymphocytes should be treated (e.g., byexposing them to IL-12) in order to enhance expression of VLA-6, anintegrin necessary for tumor infiltration (Roussel et al., 1997, J.Leuk. Biol. 62:356).

Expansion of blood lymphocytes can be achieved by collecting lymphocytesfrom a patient. These lymphocytes will normally comprise a mixture ofTh1-Tc1 and Th2-Tc2 cells. The lymphocytes are maintained for five daysin the presence of a low dose if IL-2 (e.g., 10-25 units per milliliterof medium) and a low dose (e.g., 10-100 units per milliliter of medium)of either IL-12 or IFN-g to promote Th1-Tc1 expansion. Following thisincubation, non-adherent cells are collected and expanded from 0.25million cells per milliliter to 1.0 million cells per milliliter in thesame medium. The cells are expanded several fold over the course ofabout 10 days (e.g., in AIM-V serum-free medium {GIBCO} in the presenceof the same cytokines). Differentiation of the cells is achieved byadding 10-100 units per milliliter of IL-12 or IFN-a to the medium for16-24 hours prior to injection of the cells into the patient. This IL-12(or IFN-a) treatment enhances expression of integrin VLA-6. Theresulting expanded type 1 cells are infused peri-tumorally into thepatient's body at a ratio of about 10 million to 100 million cells permilliliter of targeted tumor. The cells are infused or injected at ornear the tumor site (e.g., within a void in the tumor mass formed by atumor-antigen-release enhancing agent, such as electrical current or astrong acid, or at a plurality of sites surrounding the tumor mass).

Alternatively, lymphocytes can be expanded by incubating bloodlymphocytes obtained from a patient with T cell receptor-specificantibodies fixed to beads (e.g., polyacrylamide beads). After rinsingthe beads, they can be suspended in a medium, at a concentration ofabout 1 million beads per million cells, in a medium such as AIM-V inthe presence of 10-100 units per milliliter IL-12 or IFN-g. Cells in thesuspension are expanded several fold over the course of about 10 days.Prior to injecting the cells into the patient, the cells are separatedfrom the beads and differentiated by exposing them to 10-100 units permilliliter IFN-a for 16-24 hours in order to enhance expression ofVLA-6. The resulting expanded type 1 cells are infused peri-tumorallyinto the patient's body at a ratio of about 10 million to 100 millioncells per milliliter of targeted tumor. The cells are infused orinjected at or near the tumor site.

It is understood that in certain patients (e.g., those who mount anaggressive immune response to the tumor following the initial inductionof type 1 inflammation and those who are afflicted with only smalltumors), promotion of a sustained type 1 inflammatory response will beunnecessary. Thus, the anti-tumor method described herein need notinclude a step in which promotion of such inflammation is sustained.Omission of this step may adversely affect production of immune memorycells, resulting in a less aggressive immune response in the event thetumor recurs. Thus, this step is preferably not omitted.

Generation and Proliferation of Immune Memory Cells

The human immune system will normally produce immune memory cells duringthe course of a reaction to a pathogen-infected or tumor cell when atype 1 inflammatory response occurs in association with those cells.Production of immune memory cells can be insufficient to achieve aneffective immune response upon recurrence of the tumor. However,generation and proliferation of immune memory cells can be enhancedduring a type 1 inflammatory response by administering one or moreappropriate agents (i.e., “memory cell-inducing agents” such as IL-15and IFN-a) to the patient, preferably, at the site of inflammatoryresponse.

In one embodiment, production of immune memory cells is enhanced byadministering about 100-1000 units per milliliter of tumor volume ofIL-15 at the tumor site. Administration of IL-15 decreases the intensityof the inflammatory response and promotes conversion of activated Tcells into memory T cells. These memory T cells endure in the body andperform a ‘patrolling’ function for many years. Further growth of thetumor at the treatment site, or growth of the tumor at a distant site,can induce a rapid anti-tumor immune response, thereby providingprotection against recurrence of the tumor in the patient.

Of course, the anti-tumor treatment described herein can be performedwithout enhancing generation and proliferation of immune memory cells.However, owing to the long-term protective effect that can be achievedif this step is performed, the method preferably includes a step of thistype.

Patient Nutrition

In order to maximize the effectiveness of the anti-tumor treatment, thegeneral state of health of the patient's immune system should besupported to the greatest extent possible, so that the treatment is notlimited by immunodeficiencies not related to the patient's tumor load.For example, the patients nutritional intake should be monitored, andsupplemented if necessary, in order to ensure that nutritionaldeficiencies do not limit the capacity of the patient's immune cells tomount a type 1 inflammatory response. Examples of suitable nutritionalregimens have been described (e.g., Bendich, 1997, Nutrition,13:154-155; Weber et al., 1997, Nutrition 13:450-460; Rayman, 2000,Lancet 356:233-241; Anura et al., 1998, Am. J. Clin. Nutr.68(suppl):447S-463S). It is believed that particular nutritionalrequirements for which patient compliance should be monitored include adaily uptake of vitamin C (200-400 milligrams), vitamin E (200-400 IU),selenium (200-400 micrograms), and zinc (15-100 milligrams). Inaddition, a multi-vitamin formula that includes the vitamins A, B, andD, and minerals such as calcium, magnesium, iron, copper, and traceamounts of other vitamins and minerals normally found in awell-equilibrated formula should be included in the patient's daily dietin order to enhance the patient's immune function.

Compositions

The invention includes compositions which are useful in performance ofthe methods described herein. Such compositions can include individualagents which are packaged in a form convenient for one or more ofstorage, transportation, and administration to human patients. Thesecompositions can also include multiple agents which can be administeredto patients in the form of a single composition (e.g., multipleIR1-promoting agents, multiple leukocyte attractants, multiple antigenreleasing agents, multiple type 1 lymphocyte attractants, multiplenutritional supplements, and chemically compatible combinations of theseingredients). By way of example, a composition that is useful in thetherapeutic methods described herein comprises both a leukocyteattractant and at least one IR1-promoting agent.

Kits

The invention also includes kits that include one or more of thecompositions described herein. Various kits combine, in a single packageor in a plurality of packages that are sold, shipped, or promoted forcomplementary use, one or more of the compositions (e.g., in bulkpackages or unit dosage forms), equipment or devices (e.g., electrodesor syringes), and instructional materials described herein. Theinstructional material can be a printed material, an audio or visualmaterial, a computer-readable document or presentation, or any othertangible medium of expression whereby use of one or more of the methodsand compositions described herein is explained to a medicalpractitioner, to a patient, or both. The instructional material need notbe a single instructional material, but can be a series of pamphlets,videotapes, audio recordings, product package inserts, and the like.These materials can be provided together or separately with variouscompositions and devices described herein, or they can be providedseparately with the intention that the materials will be used inconjunction with compositions or devices described herein in order toperform one of the therapeutic methods described herein.

Medical information obtained from patients undergoing a therapydescribed herein can be assembled in various ways to make a database. Inthis database, treatment parameters (e.g., type and dose of variousagents, dosing schedule, and the like) can be correlated with thecharacteristics of the patient (e.g., age, gender, general state ofhealth), the disease being treated (e.g., with the tumor type or stageof progression), with the outcome that is achieved (e.g., the rapiditywith which the tumor is shrunk), and with any information relating torecurrence. The resulting database can be consulted when therapy of anew patient is undertaken, so that the method used to treat that patientcan be matched with therapies which resulted in a favorable outcome insimilar patients. This database can be provided (e.g., in the form of acomputer-readable database) to users of the compositions, kits, andmethods described herein, or it can be consulted by experts whocommunicate with those users.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention is not limited to these Examples, but rather encompass allvariations which are evident as a result of the teaching providedherein.

Example 1

A patient diagnosed with a breast tumor mass having a diameter of 5centimeters is treated using the anti-tumor treatment described herein,as follows.

A 500 milliliter sample of the patient's peripheral blood is withdrawn,and the cells are expanded in vitro, using a method described in theDetailed Description or in the prior art. The patient is instructed tobegin taking a multi-vitamin formula including daily amounts of 300milligrams of vitamin C, 300 IU of vitamin E, 200 micrograms ofselenium, and 50 milligrams of zinc. The total dosage of the supplementis divided in three equal doses, one to be taken with each of the threedaily meals.

After five days, the patient is prepared for tumor de-bulking by beingimmobilized in a bed and given a local anesthetic to desensitize thenerves of the breast tissue. With the assistance of live digitalimaging, one electrode (negative polarity) is inserted in the center ofthe tumor mass, and two electrodes (each positive polarity) areinserted, one in each end of the tumor, about 2 centimeters from thecenter. Electric current is activated, and performed as described in theDetailed Description, for a duration of 2 hours. Thereafter, the patientrests for six hours.

In order to promote tumor leukocyte infiltration, a suspensioncomprising human IFN-g, TFN-a, IP-10, and Mig, each at a concentrationspecified in the Detailed Description. The suspension is injected atfive points distributed equally at the tumor periphery, each injectioncontaining about twenty microliters of the suspension. The patientremains at rest for 24 hours.

The patient is injected with an inflammation polarizing mixturecomprising IFN-g, TNF-b, and IL-2, each at a concentration specified inthe Detailed Description. This polarizing mixture is injected twicemore, at 48 and 96 hours after the initial injection.

One hour later, about five billion expanded peripheral blood lymphocytes(expanded from the patient's blood sample) are injected peri-tumorally,at five different sites surrounding the tumor. An injection of theexpanded cells is made into each of the three holes created by theelectrical de-bulking treatment, and two more injections are made in theperiphery of the tumor. An aliquot of the suspension comprising IFN-g,IL-2, and TNF-a is injected intra-tumorally (although this suspensioncan, alternatively, be combined with the cell suspension prior toperi-tumoral infusion of the cells). This amplification step is repeatedevery two or three days until the tumor has regressed to about 10percent of its original size.

IL-15 is injected at the tumor site, as described in the DetailedDescription, in order to terminate the type 1 inflammation by convertingactivated T cells into memory cells. The patient is thereafterdischarged, and monthly follow-up sessions are scheduled.

Example 2

A patient in a poor nutritional state caused by a wide, thin stomachtumor covering the right wall of his stomach is treated using theanti-tumor treatment described herein.

The patient is placed on intravenous nutrition, including the dailynutritional requirements described in the Detailed Description. Thistreatment is continued for six days in order to partially replenish thepatient's nutritional state.

Hexadecylphosphocholine (in an amount described in the DetailedDescription) is injected at a plurality of points within the tumor, theinjections being guided by live digital imaging. A mixture of proteases,as described in the Detailed Description, is injected at a plurality ofpoints in the tumor. The inflammation at the tumor site is monitoredafter 24 hours, and the tumor should exhibit much infiltration andinflammation, as is characteristic of stomach tumors.

A suspension comprising IFN-g, TNF-b, and IL-2, in amounts specified inthe Detailed Description, is injected at several points in the tumormass in order to polarize the inflammatory response to a type 1 responsein the tumor tissue. The tumor is monitored daily, and the polarizingsuspension is injected into the tumor every 48 hours. After five days onthis regimen, the tumor is anticipated to regress at a rate of about 10percent of its total surface per day. After 8 more days, the tumor isanticipated to have shrunk to about 15 percent of its original size, butthe tissues surrounding the tumor are anticipated to have becomeinflamed, and the patient experiences stomach pain.

The polarization treatment is discontinued, and IFN-a is injected intothe tumor mass in order to halt the inflammatory response and inducememory cell production. IFN-a is also injected into the inflamed tissuesurrounding the remaining tumor every two or three days, until the tumorhas substantially disappeared, and the inflammation has diminishedconsiderably.

Oral nutritional intake can then be re-established in the patient. Oncethis is re-established, the patient is discharged and monthly follow-upmonitoring sessions are scheduled.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention can be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims include all such embodiments and equivalent variations.

1. A method of inducing tumor cell death in a human patient by inducinga type 1 inflammatory response in a solid tumor, the method comprisingi) locally administering an antigen-releasing agent to a solid tumor inthe patient, whereby a tumor antigen is released from cells of thetumor; iia) locally administering to the tumor a leukocyte attractant,whereby leukocytes are induced to infiltrate the tumor; and iib) locallyadministering to the tumor interferon-gamma (IFN-g) as a first tyne 1inflammatory response promoting agent and a second type 1 inflammatoryresponse-(IR1-)promoting agent selected from the group consisting oftumor necrosis factor-beta (TNF-b), tumor necrosis factor-alpha (TNF-a),interleukin-2 (IL-2), interleukin-12 (IL-12), and a mixture thereofwhereby a type 1 inflammatory response is induced in the tumor and tumorcell death is induced.
 2. The method of claim 1, wherein theantigen-releasing agent is a tumor de-bulking agent.
 3. The method ofclaim 1, wherein the antigen-releasing agent comprises an agent selectedfrom the group consisting of a proteolytic enzyme, an apoptosis-inducingagent, electrical current, an acid selected from the group consisting ofhydrochloric acid and sulfuric acid, a base selected from the groupconsisting of sodium hydroxide and potassium hydroxide; and a mixturethereof.
 4. The method of claim 3, wherein the antigen-releasing agentcomprises a proteolytic enzyme selected from the group consisting oftrypsin, chymotrypsin, pepsin, collagenase, and a mixture thereof. 5.The method of claim 3, wherein the antigen-releasing agent comprisesonly one proteolytic enzyme.
 6. The method of claim 3, wherein theantigen-releasing agent comprises at least two proteolytic enzymes. 7.The method of claim 3, wherein the antigen-releasing agent comprises analkylphospholipid.
 8. The method of claim 7, wherein thealkylphospholipid is an alkylphosphocholine.
 9. The method of claim 7,wherein the alkylphosphocholine is selected from the group consisting ofhexadecylphosphocholine, edelfosine, and a mixture thereof.
 10. Themethod of claim 3, wherein the antigen-releasing agent is electricalcurrent delivered by way of electrodes inserted into the tumor.
 11. Themethod of claim 3, wherein the antigen-releasing agent comprises an acidselected from the group consisting of hydrochloric acid, sulfuric acid,and a mixture thereof.
 12. The method of claim 3, wherein theantigen-releasing agent comprises a base selected from the groupconsisting of sodium hydroxide, potassium hydroxide, and a mixturethereof.
 13. The method of claim 1, wherein the antigen-releasing agentis administered to the tumor at least two hours before administering theleukocyte attractant to the tumor.
 14. The method of claim 1, whereinthe antigen-releasing agent and the leukocyte attractant areco-administered to the tumor.
 15. The method of claim 1, wherein theantigen-releasing agent is administered to the tumor at least two hoursbefore administering IFN-g to the tumor.
 16. The method of claim 1,wherein the antigen-releasing agent and the IFN-g are co-administered tothe tumor.
 17. The method of claim 1, wherein the leukocyte attractantcomprises a monocyte attractant.
 18. The method of claim 17, wherein themonocyte attractant is selected from the group consisting of MCP-1,MCP-2, MCP-3, and MCP-4, and a mixture thereof.
 19. The method of claim1, wherein the leukocyte attractant comprises a T cell attractant. 20.The method of claim 19, wherein the T cell attractant is selected fromthe group consisting of RANTES, IP-10, and Mig, and a mixture thereof.21. The method of claim 1, wherein the leukocyte attractant comprises agranulocyte attractant.
 22. The method of claim 21, wherein thegranulocyte attractant is selected from the group consisting ofinterleukin-8, granulocyte chemotactic protein-2, growth-relatedoncogen-1, growth-related oncogen-2, growth-related oncogen-3,neutrophil activated protein, neurotactin, and a mixture thereof. 23.The method of claim 21, wherein the granulocyte attractant is aeosinophil attractant.
 24. The method of claim 23, wherein theeosinophil attractant is eotaxin.
 25. The method of claim 1, wherein theleukocyte attractant is co-administered with at least one of IFN-g andthe second IR1-promoting agent.
 26. The method of claim 1, wherein theleukocyte attractant and at least one of IFN-g and the secondIR1-promoting agent are administered not more than two hours apart. 27.The method of claim 1, wherein the leukocyte attractant and at least oneof IFN-g and the second IR1-promoting agent are administered more thantwo hours apart.
 28. The method of claim 1, wherein the leukocyteattractant and at least one of IFN-g and the second IR1-promoting agentare co-administered.
 29. The method of claim 1, which further comprisesadministering at least one additional IR1-promoting agent is selectedfrom the group consisting of interleukin-2 (IL-2), interleukin-12(IL-12), tumor necrosis factor-alpha (TNF-a), and tumor necrosisfactor-beta (TNF-b) and a mixture thereof.
 30. The method of claim 29,wherein multiple aliquots of each of IFN-g , the second and theadditional IR1-promoting agents are administered to the patient, andwherein at least 48 hours elapse between aliquots.
 31. The method ofclaim 29, wherein IFN-g, the second and the additional IR1-promotingagents are co-administered.
 32. The method of claim 29, wherein IFN-g,the second and the additional IR1-promoting agents are separatelyadministered not more than two hours apart.
 33. The method of claim 29,wherein IFN-g, the second and the additional IR1-promoting agents areseparately administered more than two hours apart.
 34. The method ofclaim 1, further comprising iii) locally administering to the tumor atype 1 lymphocyte attractant in order to sustain the type 1 inflammatoryresponse.
 35. The method of claim 34, wherein the type 1 lymphocyteattractant is selected from the group consisting of RANTES, IP-10, Mig,and a mixture thereof.
 36. The method of claim 34, wherein two type 1lymphocyte attractants are locally administered and wherein the type 1lymphocyte attractants are IP-10 and Mig.
 37. The method of claim 34,further comprising iv) sustaining the type 1 inflammatory response bylocally administering autologous leukocytes to the tumor.
 38. The methodof claim 34, further comprising iv) administering a memory cell-inducingagent to the patient after inducing the type 1 inflammatory response,whereby production of anti-tumor type 1 immune memory cells is enhanced.39. The method of claim 38, further comprising v) supplementing thepatient's nutrition with a nutrient selected from the group consistingof a vitamin, a mineral, and a mixture thereof.
 40. The method of claim34, further comprising iv) supplementing the patient's nutrition with anutrient selected from the group consisting of a vitamin, a mineral, anda mixture thereof.
 41. The method of claim 1, further comprising iii)locally administering autologous leukocytes to the tumor.
 42. The methodof claim 41, wherein the autologous leukocytes are obtained from thepatient and expanded prior to locally administering them to the tumor.43. The method of claim 41, wherein the autologous leukocytes areobtained from the patient and contacted with one or more IR1-promotingagent prior to locally administering them to the tumor.
 44. The methodof claim 41, wherein the autologous leukocytes are obtained from thepatient, expanded ex vivo, and contacted with one or more IR1-promotingagent prior to locally administering them to the tumor.
 45. The methodof claim 44, wherein the leukocytes are contacted with both the one ormore IR1-promoting agent and with at least one of interferon-alpha(IFN-a) and IL-12 prior to locally administering them to the tumor. 46.The method of claim 41, further comprising iv) administering a memorycell-inducing agent to the patient after inducing the type 1inflammatory response, whereby production of anti-tumor type 1 immunememory cells is enhanced.
 47. The method of claim 46, further comprisingv) supplementing the patient's nutrition with a nutrient selected fromthe group consisting of a vitamin, a mineral, and a mixture thereof. 48.The method of claim 41, further comprising iv) supplementing thepatient's nutrition with a nutrient selected from the group consistingof a vitamin, a mineral, and a mixture thereof.
 49. The method of claim1, further comprising iii) administering a memory cell-inducing agent tothe patient after inducing the type 1 inflammatory response, wherebyproduction of anti-tumor type 1 immune memory cells is enhanced.
 50. Themethod of claim 49, wherein the memory cell-inducing agent is selectedfrom the group consisting of interleukin-15 (IL-15), IFN-a, and amixture thereof.
 51. The method of claim 49, wherein the memorycell-inducing agent is IL-15.
 52. The method of claim 49, wherein thememory cell-inducing agent is IFN-a.
 53. The method of claim 49, whereinthe memory cell-inducing agent is administered after the tumor shrinksto less than 10 percent of its size, immediately prior to administrationof the memory cell inducing agent.
 54. The method of claim 1, furthercomprising supplementing the patient's nutrition with a nutrientselected from the group consisting of a vitamin, a mineral, and amixture thereof.
 55. The method of claim 54, wherein the vitamin isselected from the group consisting of vitamins A, B, C, D, E, and amixture thereof.
 56. The method of claim 55, wherein the vitamin isvitamin C and wherein the patient's nutrition is supplemented such thatthe patient receives from 200 to 400 milligrams of vitamin C daily. 57.The method of claim 55, wherein the vitamin is vitamin E and wherein thepatient's nutrition is supplemented such that the patient receives from200 to 400 international units of vitamin E daily.
 58. The method ofclaim 54, wherein the mineral is selected from the group consisting ofselenium, zinc, calcium, magnesium, iron, copper, and a mixture thereof.59. The method of claim 58, wherein the mineral is selenium and whereinthe patient's nutrition is supplemented such that the patient receivesfrom 200 to 400 micrograms of selenium daily.
 60. The method of claim58, wherein the mineral is zinc and wherein the patient's nutrition issupplemented such that the patient receives from 15 to 100 milligrams ofzinc daily.
 61. The method of claim 54, wherein the patient's nutritionis supplemented beginning at least on the same day that theantigen-releasing agent is administered to the tumor, and continuingthrough at least the same day that IFN-g is administered to the tumor.62. The method of claim 54, wherein the patient's nutrition issupplemented beginning at least five days before the antigen-releasingagent is administered to the tumor, and continuing through at leastthree days after the day that IFN-g is administered to the tumor.
 63. Amethod of inducing tumor cell death in a human patient by inducina atype 1 inflammatory response in a solid tumor, the method comprising i)supplementing the patient's nutrition with a nutrient selected from thegroup consisting of a vitamin, a mineral, and a mixture thereof; ii)locally administering an antigen-releasing agent to a solid tumor in thepatient, whereby a tumor antigen is released from cells of the tumor;thereafter iiia) locally administering to the tumor a leukocyteattractant, whereby leukocytes are induced to infiltrate the tumor; andiiib) locally administering to the tumor interferon-gamma (IFN-g) as afirst type 1 inflammatory response promoting agent and a second type 1inflammatory response-(IR1-)promoting agent, selected from the groupconsisting of tumor necrosis factor-beta (TNF-b), tumor necrosisfactor-alpha (TNF-a), interleukin-2 (IL-2), interleukin-12 (IL-12), anda mixture thereof whereby a type 1 inflammatory response is induced inthe tumor; thereafter iv) sustaining the type 1 inflammatory response byiva) locally administering to the tumor a type 1 lymphocyte attractant,ivb) locally administering autologous leukocytes to the tumor, or ivc)both iva) and ivb); and thereafter v) administering a memorycell-inducing agent to the patient after inducing the type 1inflammatory response, whereby production of anti-tumor type 1 immunememory cells is enhanced and tumor cell death is induced.
 64. A methodof inducing a type 1 inflammatory response at the site of a solid tumorin a human patient, the method comprising locally co-administering tothe tumor p1 i) an antigen-releasing agent; ii) a leukocyte attractant;and iii) interferon-gamma (IFN-g) as a first type 1 inflammatoryresponse promoting agent and a second type 1 inflammatoryresponse-(IR1-) promoting agent selected from the group consisting oftumor necrosis factor beta (TNF-b), tumor necrosis factor-alpha (TNF-a),interleukin-2 (Il -2), interleukin-12 (Il -12), and a mixture thereof.